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Gene chip and its detection method for identification of six kinds of swine disease pathogens

A technology of gene chips and pathogens, applied in the field of biochips, can solve the problem of lack of parallel detection of six pathogens, and achieve the effect of strong and stable hybridization signal, high sensitivity and strong stability

Active Publication Date: 2019-03-08
重庆海关技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, gene chip detection technology has been applied to the detection of many diseases, but there is a lack of parallel detection technology for six pathogens

Method used

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  • Gene chip and its detection method for identification of six kinds of swine disease pathogens
  • Gene chip and its detection method for identification of six kinds of swine disease pathogens
  • Gene chip and its detection method for identification of six kinds of swine disease pathogens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1, the present invention respectively cultures Porcine Infectious Actinobacillus pleuropneumoniae, Haemophilus parasuis and Mycoplasma hyopneumoniae, and extracts pathogen genome DNA; Porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and CSFV cells were propagated, and the virus genome was extracted. Cloning, sequencing and analysis of the specific conserved sequences of Actinobacillus pleuropneumoniae, Haemophilus parasuis, Mycoplasma hyopneumoniae, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and classical swine fever virus, according to the sequence Results Six oligonucleotide probes were designed, which could specifically bind to the PCR products of corresponding pathogens. When performing genome amplification by PCR, the 5′ end of the upstream universal primer was labeled with Cy3 fluorescent dye, and the probes could bind to the PCR products. Hybridization was performed at 42°C. According t...

Embodiment 2

[0081] Example 2, see figure 1 , the gene chip used for the detection of important pathogens of respiratory disease syndrome, adopts the micro-spotting technology of the gene chip, immobilizes the probes of the samples to be tested and various quality control probes on the chemically modified substrate, forming 8 rows × 7 columns microarray. The distribution of probes on the microarray from top to bottom is as follows: spotting position quality control partition P1: 1 row × 8 points, hybridization positive quality control partition P2: 1 row × 4 points, hybridization negative quality control partition N: 1 row × 4 o'clock, porcine infectious Actinobacillus pleuropneumoniae detection probe division 1: 1 row × 4 dots, Haemophilus parasuis detection probe division 2: 1 row × 4 dots, Mycoplasma hyopneumoniae detection probe division 3: 1 Row × 4 dots, porcine circovirus type 2 detection probe division 4: 1 row × 4 dots, porcine reproductive and respiratory syndrome virus detectio...

Embodiment 3

[0084] Embodiment 3, reagent preparation

[0085] 1) Chip washing solution

[0086] According to needs and actual conditions, prepare washing solution I and washing solution II according to the following proportions.

[0087] Washing solution Ⅰ: the final concentration of SSC is 2×, and the final concentration of SDS is 0.2%. For example, 600 mL of washing liquid Ⅰ=528 mL of distilled water+60mL of 20×SSC+12mL of 10%SDS, or prepare in proportion as required.

[0088] Washing solution Ⅱ: The final concentration of SSC is 0.2×. For example, 600 mL of washing solution II=594 mL of distilled water+6 mL of 20×SSC, or prepare in proportion as required.

[0089] If 10% SDS produces a white flocculent precipitate, please dissolve it in a water bath at 42°C and mix well to prepare a lotion.

[0090] 2) Absolute ethanol.

[0091] 3) Ice-water mixture.

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Abstract

The invention discloses a gene chip for identification of six swine disease pathogens and a detection method thereof. The gene chip is used for detection of porcine actinobacillus pleuropneumoniae, haemophilus parasuis, mycoplasma hyopneumoniae, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and classical swine fever virus. A PCR primer is designed by means of standard strain genome sequence analysis, cloning and sequencing analysis are carried out on a target gene, and specific probes are designed to perform identification detection on porcine actinobacillus pleuropneumoniae, haemophilus parasuis, mycoplasma hyopneumoniae, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and classical swine fever virus 6 pathogens at the same time. The invention aims to establish the microarray chip with the advantages of high sensitivity, strong specificity, time saving and labor saving, and easy observation of results to identify and detect the important pathogens of porcine respiratory disease complex.

Description

technical field [0001] The invention belongs to the field of biological chips. Specifically, the present invention relates to a differential detection of six pathogens of porcine infectious Actinobacillus pleuropneumoniae, Haemophilus parasuis, Mycoplasma hyopneumoniae, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and classical swine fever virus The chip device and its detection method. Background technique [0002] Mycoplasma pneumoniae of swine (MPS), porcine contagious pleuropneumonia (PCP), swine polyserositis and arthritis, porcine circovirus associated disease , PCVAD), porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) and classical swine fever (CSF) are caused by Mycoplasma hyopneumoniae (Mhp), porcine infectious Actinobacillus pleuropneumoniae ( Actinobacillus pleuropneumoniae, APP), Haemophilus parasuis (Hps), porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syn...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/689C12Q1/6837C12Q1/04C12R1/21C12R1/35C12R1/93C12R1/01
CPCC12Q1/6837C12Q1/689C12Q1/701C12Q2531/113C12Q2565/501
Inventor 李应国王昱聂福平保雨杨俊艾军张强王国民
Owner 重庆海关技术中心
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